Biochips or biological microarrays, in particular DNA microarrays, have become an important tool in modern molecular biology and medicine. Typically the chips consist of an arrayed series of a large number of microscopic spots of nucleic acid molecules, each containing small amounts of a specific nucleic acid sequence. This can be, for example, a short section of a gene or other DNA element that are used as capture probes to hybridize a cDNA or cRNA sample (a target or target probe) under conditions, which allow a binding between the capture probe and the corresponding target. Capture probe-target hybridization is typically detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment. The use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon. These early gene arrays were made by spotting cDNAs onto filter paper with a pin-spotting device. The use of miniaturized microarrays, in particular for gene expression profiling was first reported in the 1990s. A complete eukaryotic genome on a microarray was published in 1997.
A variety of technologies may be used in order to fabricate such microarrays. The techniques include printing with fine-pointed pins, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing (Lausted C et al., 2004, Genome Biology 5: R58), or electrochemistry.
The photolithographic technique is directed to the production of oligonucleotide arrays by synthesizing the sequences directly onto the array surface. The technique involves photolithographic synthesis on a silica substrate where light and light-sensitive masking agents are utilized to generate a sequence one nucleotide at a time across the entire array (Pease et al., 1994, PNAS 91: 5022-5026). Each applicable probe is selectively unmasked prior to bathing the array in a solution of a single nucleotide, then a masking reaction takes place and the next set of probes are unmasked in preparation for a different nucleotide exposure. After several repetitions, the sequences of every probe become fully constructed. Accordingly constructed oligonucleotides may be longer (e.g. 60-mers) or shorter (e.g. 25-mers) depending on the desired purpose.
In spotted microarrays, the oligonucleotide probes are deposited as intact sequences, i.e. the probes are synthesized prior to deposition on the array surface and are then spotted onto the substrate. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface, or an ink jet printing device, which deposits the probe material via the ejection of droplets. The resulting array of probes represents the nucleic acid profiles of the prepared capture probes and can interact with complementary cDNA or cRNA target probes, e.g. derived from experimental or clinical samples. In addition, these arrays may be easily customized for specific experiments, since the probes and printing locations on the arrays can be chosen specifically.
Central to several of these techniques is the efficient immobilization of the nucleic acid onto the support or material. A classical way of immobilizing nucleic acids on a support is the application of UV-light, i.e. UV-crosslinking. Church and Gilbert described 1984 that the application of UV light of a wavelength of 254 nm leads to the immobilization of DNA fragments to nylon filters (Church and Gilbert, PNAS, 1984, 81, p: 1991-1995). An advanced version of this approach was proposed by Saiki et al., PNAS, 1989, 86, p: 6230-6234, which described that oligonucleotides comprising poly(dT) tails could more readily be fixated to membranes when using UV-light of a wavelength of 254 nm. The effect was attributed to a more efficient binding of light-activated thymine bases to a membrane.
However, irradiating nucleic acids with short wavelength UV-light of around 254 nm typically leads to severe damages to the nucleic acid molecule, which may have consequences for the capability of the molecules to hybridize with complementary sequences and could thus compromise their usability in microarray systems. In particular, such an irradiation may lead to the generation of pyrimidine dimers due to the establishment of cyclobutan rings between adjacent pyrimidines, in particular between neighboring thymine residues. A further effect may be the generation of associations between adjacent thymine and cytosine residues, resulting in TC(6,4)-products.
There is, thus, a need for an alternative immobilization method for nucleic acids which allows an efficient fixation of the nucleic acid on a support that overcomes the disadvantageous damages to the molecules accompanied with a traditional UV-crosslinking at around 254 nm.